Novel signal molecules, such as the free radical NO, participate in intra- and intercellular communication. NO is produced by NOS isoenzymes of at least three major classes and inflammatory cytokines upregulate the iNOS in such cells as macrophages, which share a hematopoietic origin with osteoclasts. Although NO inhibits osteoclast bone resorption, initial studies indicate that osteoclasts, like osteoblasts, stromal cells and endothelial cells, may produce NO. The test hypothesis in this proposal is that NO may act, both in an autocrine and paracrine fashion, to influence osteoclast function. Moreover, it is suggested that changes in NOS isoform expression may be a function of osteoclast development. The application's Specific Aims are to: (1) clone and identify avian osteoclast NOS(s) using a combination of RT-PCR and lambda ZapII library cloning techniques, DNA sequencing, in situ hybridization and antibody detection; (2) evaluate the potential regulation of autocrine NO actions of osteoclasts by monitoring changes in osteoclast activity, NO production and NOS isoform expression in response to selected modulators of osteoclast activity such as IL-1, estradiol, glucocorticoids, superoxide or bone matrix. Osteoclast function will be assessed using the isolated avian osteoclast bone pit resorption assay, changes in lysosomal enzyme activity and alterations in osteoclast 121F antigen expression; and (3) analyze whether developmental changes occur in NOS expression during osteoclast cytodifferentiation using a) marrow mononuclear cells cultured to form osteoclast-like giant cells, and b) the chick chorioallantoic membrane (CAM) implant system, in which mature resorptive osteoclasts develop from circulating precursors. In situ hybridization, PCR analysis, immunodetection, and quantitative RNAase protection assays will be utilized to evaluate the NOS system during osteoclast differentiation. These studies are intended to contribute to a better understanding of the cellular and molecular biology of osteoclast NO in normal and pathological bone remodeling.